source: Human oral microbiome from patients with advanced periodontitis
Extracted molecule
genomic DNA
Extraction protocol
For oral microbiome sampling, bacteria were separated from the paper-points by vortexing. The paper points were discarded and community DNA was extracted using the QIAamp TM DNA micro Kit (QIAGEN Sciences, Maryland, USA) following the manufacturer’s instructions and adding a lysozyme (3 mg/mL, 1.5 h) treatment step. For gut microbiome sampling, all fecal samples were immediately frozen on collection and stored at -70℃ before analysis. A frozen aliquot (200 mg) of each fecal sample was added to a 2.0-ml screwcap vial containing 300 mg glass beads of 0.1 mm diameter (Sigma, St. Louis, MO, USA), and kept on ice until the addition of 1.4-ml ASL buffer from the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA, USA). Samples were immediately subjected to beadbeating (45 s, speed 6.5) using a FastPrep machine (Bio 101, Morgan Irvine, CA, USA), prior to the initial incubation for heat and chemical lysis at 95 ℃ for 5 minutes. Subsequent steps of DNA extraction followed the QIAamp kit protocol for pathogen detection.
Label
Cy5
Label protocol
DNA was labeled with the fluorescent dye Cy5 (GE Healthcare) by random priming
Hybridization protocol
DNA was suspended in hybridization buffer and then kept at 65°C until hybridization. Hybridizations were performed at 42°C for 10 h using a Tecan HS4800 Pro hybridization station
Scan protocol
Arrays were scanned with a ScanArray 500 microarray scanner at 633 nm using a laser power of 90% and a photomultiplier tube (PMT) gain of 75%.
Data processing
Signal intensity were normalized by CORS universal standard probes (Liang et. al 2010 AEM), probes with SNR<2 are removed, normalized signal intensities was calculated for further statistical analysis. We used minimal signal intensity of 1000 and SNR (Signal to Noise Ratio) cutoff of 2 for positive callings of the presence of a gene.
